We have previously demonstrated that multiple immunizations with vector-based vaccines containing transgenes for tumor antigens (Ags) and a triad of costimulatory molecules (TRICOM) enhance the expansion and functional avidity of Ag-specific memory CD8+ T cells in a mouse model. However, the effect of enhanced costimulation on human memory CD8+ T cells is still unclear. We conducted in vitro investigation of the proliferation and function of CEA-specific human memory CD8+ T cells following enhanced costimulation. Our results demonstrated that TRICOM costimulation enhanced production of multiple cytokines and expansion of CEA-specific memory CD8+ T cells. The lytic capacity of memory cytotoxic T lymphocytes (CTLs) toward CEA+ tumors was also significantly enhanced. IL-2Ralpha (CD25) was upregulated dramatically following APC (antigen-presenting cell)-TRICOM stimulation, suggesting that the enhanced expansion of memory CD8+ T cells may be mediated by increased expression of IL-2R on memory T cells. The enhanced cytokine production and proliferation following TRICOM signaling was completely blocked by the combination of neutralizing Abs against B7-1, ICAM-1, and LFA-3, the costimulatory molecules comprising TRICOM. No difference in T-cell apoptosis was observed between APC-TRICOM and APC-wild-type groups, as determined by annexin V, Bcl-2, and active caspase-3 staining. Results indicated that enhanced costimulation greatly expanded human CEA-specific CD8+ T cells and enhanced T-cell function, without inducing increased apoptosis of CEA-specific memory CD8+ T cells.We have previously shown that the suppressive function of regulatory T cells (Tregs) from peripheral blood mononuclear cells (PBMCs) is enhanced in patients with prostate cancer when compared with healthy individuals. Two phase II studies using the PSA-TRICOM vaccine in patients with metastatic castration-resistant prostate cancer (mCRPC) showed evidence of patient benefit in terms of enhanced survival. The Halabi nomogram has been used to predict survival (HPS) of patients with mCRPC treated with conventional chemotherapy or second-line hormonal therapy. Tregs from PBMCs of patients (n = 23) with mCRPC were obtained pre- and post-three monthly vaccinations, and analyzed for number, phenotype, and suppressive function. Changes post- versus pre-vaccination in these parameters were compared with 3-year survival and HPS. No differences in Treg numbers were observed post- versus prevaccination. Trends (P = 0.029) were observed between overall survival (OS) and a decrease in Treg suppressive function post- versus pre-vaccination. Trends were also observed in analyzing effector:Treg (CD4+CD25+CD127-FoxP3+CTLA4+) ratio post- versus pre-vaccination with OS versus HPS. These data provide preliminary evidence for a possible association between improved OS and a decrease in Treg function when PBMCs are analyzed after three monthly vaccinations. Patients with an OS greater than HPS were more likely to have decreased Treg function following vaccine. Larger studies to confirm and extend these findings are warranted.We have compared the effects of recombinant Saccharomyces cerevisiae (yeast)-treated human dendritic cells (DCs) with CD40L-matured human DCs for the induction of effector cells and the number and functionality of CD4+CD25+CD127-FoxP3+ regulatory T cells (Tregs). DCs were treated with yeast or CD40L and cocultured with isolated autologous CD4+ T cells. CD4+CD25+CD127- T cells isolated from the coculture of CD4+ T cells plus yeast-treated DCs (yeast coculture) had a lower expression of FoxP3 and decreased suppressive function compared to CD4+CD25+CD127- T cells isolated from the coculture of CD4+ T cells plus CD40L-treated DCs (CD40L coculture). Also, compared to the CD40L coculture, the yeast coculture showed increases in the ratio of CD4+CD25+ activated T cells to Tregs and in the production of Th1-related cytokines (IL-2, TNF-alpha, IFN-gamma) and IL-6. In addition, yeast-treated DCs used as APCs incubated with the tumor antigen CEA enhanced the proliferation of CEA-specific CD4+ T cells compared to the use of CD40L-matured DCs used as APCs. This is the first study to report on the role of yeast-treated/matured human DCs in reducing Treg frequency and functionality and in enhancing effector to Treg ratios. These results provide an additional rationale for the use of yeast as a vector in cancer vaccines.